Identification of potential genes/enzymes involved in biosynthesis pathway of polyacetylenes in Bidens pilosa L.

Bidens pilosa L. (BP) from family Asteraceae is a cosmopolitan annual medicinal herb, known for its traditional use in treating various diseases. Polyacetylenes have been identified from B. pilosa plants showing anti-angiogenesis, anti-type I diabetes, and other bioactivities. The bioactive polyacetylenic compounds contain a unique carbon–carbon triple or double bond functionality, however, their biosynthesis in BP plant is still unknown. Previous study in our laboratory showed that methyl jasmonate (MeJA) treatment can affect polyacetylenes accumulation in BP leaves, and transcriptomics study of BP leaves with or without MeJA treatment have been completed. The objective of this study was to identify and analyze putative enzyme genes involved in polyacetylenes biosynthesis. We used the four weeks old BP seedlings to treat with MeJA and harvested the leaf and root tissues at different time points from 0 to 16 days, and the chemical profiling and contents of six major polyacetylenes were examined by high-performance liquid chromatography (HPLC). The comparative functional genomics study of the BP leaf and root tissues, with or without treating with MeJA was used the in-house designed DNA microarray chips based on the RNA-seq data containing 39,202 annotated genes to study the gene expression profiles , aiming to select out the “tool-box” genes involved in the PAs biosynthesis. The expression profiles of selected candidate genes were further confirmed by real time RT-PCR and RNAi gene knockdown experimental system through Agrobacterium biotransformation in BP plants, in parallel, the PA profiles in the control mock plant and specific gene knockdown plant tissues were compared. Our results showed that the expression profile of four genes encoded for Δ12-oleate desaturase (BPOD), Δ12-fatty acid acetylenase (BPFAA), microsomal oleate desaturase FAD2-5 (B26081) and lipase (B35957) have similar trend to polyacetylenes production profile, which were selected to perform RNAi experiment in BP plants for their function validation. The BPOD, BPFAA and B35957 genes were revealed significant reduction in their expression levels at day 6 in concomitant to the significantly decreased of PAs accumulation in BP leaf, while B26081 gene expression level was decreased at day 3 and day 6 corresponding to the significantly decreased of PA contents at the same days. In summary, the current study demonstrates an approach to investigate the biosynthesis pathway of bioactive compound in medicinal plant. The results indicate that MeJA can potentially induce specific enzyme genes involved in polyacetylenes production in Bidens pilosa plant. Further functional validation of the selected candidate genes would anticipate to help understanding the biosynthesis pathway of bioactive polyacetylenes compounds in the medicinal plant.